Methodological approaches to help unravel the intracellular metabolome of Bacillus subtilis

Background: Bacillus subtilis (B. subtilis) has become widely accepted as a model organism for studies on Gram-positive bacteria. A deeper insight into the physiology of this prokaryote requires advanced studies of its metabolism. To provide a reliable basis for metabolome investigations, a validated experimental protocol is needed since the quality of the analytical sample and the final data are strongly affected by the sampling steps. To ensure that the sample analyzed precisely reflects the biological condition of interest, outside biases have to be avoided during sample preparation. Results: Procedures for sampling, quenching, extraction of metabolites, cell disruption, as well as metabolite leakage were tested and optimized for B. subtilis. In particular the energy status of the bacterial cell, characterized by the adenylate energy charge, was used to evaluate sampling accuracy. Moreover, the results of the present study demonstrate that the cultivation medium can affect the efficiency of the developed sampling procedure. Conclusion: The final workflow presented here allows for the reproducible and reliable generation of physiological data. The method with the highest qualitative and quantitative metabolite yield was chosen, and when used together with complementary bioanalytical methods (i.e., GC-MS, LC-MS and H-1-NMR) provides a solid basis to gather information on the metabolome of B. subtilis.